Faecalibacterium prausnitzii and Desulfovibrio piger for use in the treatment or prevention of diabetes and bowel diseases

ABSTRACT

The present invention relates generally to medicine. More specifically the invention relates to the use of synergistic probiotic bacteria as intervention for health. In particular, the present invention provides a strain of  Faecalibacterium prausnitzii  and a bacterial strain which has one or more of the characteristics of: (i) being acetate producing, (ii) being lactate consuming and (iii) having the ability to be an electron acceptor, for use in the treatment or prevention of a disease associated with reduced butyrate levels or a disease associated with reduced or low numbers of  Faecalibacterium prausnitzii  bacteria.

STATEMENT OF PRIORITY

This application is a 35 U.S.C. § 371 national phase application ofInternational Application Serial No. PCT/EP2016/076038, filed Oct. 28,2016, which claims the benefit, under 35 U.S.C. § 119 (a) of GreatBritain Patent Application No. 1519088.7, filed Oct. 28, 2015, theentire contents of each of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to medicine. More specificallythe invention relates to the use of synergistic probiotic bacteria asintervention for health. The invention discloses a method that supportsthe growth and colonization in humans of a strain of the speciesFaecalibacterium prausnitzii.

BACKGROUND OF THE INVENTION

Within the body of a healthy adult, microbial cells are estimated tooutnumber human cells by a factor of ten to one. These communities,however, remain largely unstudied, leaving almost entirely unknown theirinfluence upon human development, physiology, immunity, nutrition andhealth.

Traditional microbiology has focused on the study of individual speciesas isolated units. However many, if not most, have never beensuccessfully isolated as viable specimens for analysis, presumablybecause their growth is dependent upon a specific microenvironment thathas not been reproduced experimentally. Among those species that havebeen isolated, analyses of genetic makeup, gene expression patterns, andmetabolic physiologies have rarely extended to inter-speciesinteractions or microbe-host interactions. Advances in DNA sequencingtechnologies have created a new field of research, called metagenomics,allowing comprehensive examination of microbial communities, even thosecomprised of uncultivable organisms. Instead of examining the genomes ofindividual bacterial strains that have been cultured in a laboratory,the metagenomic approach allows analysis of genetic material derivedfrom complete microbial communities harvested from natural environments.For example, the gut microbiome complements our own genome withmetabolic functions that affects human metabolism and may thus play animportant role in health and disease.

It is believed the change of these bacteria in the intestines play arole in many chronic and degenerative diseases. There is a growing bodyof evidence that substantiates and clarifies what is called “thedysbiosis theory”.

The term “dysbiosis” was originally coined by the Nobel laureate EliMetchnikoff to describe altered pathogenic bacteria in the gut.Dysbiosis has by some been defined as “qualitative and quantitativechanges in the intestinal flora, their metabolic activity and theirlocal distribution.” Thus dysbiosis is a state, often associated withreduced microbial diversity, in which the microbiota produces harmfuleffects via, for example:

-   -   qualitative and quantitative changes in the intestinal flora        itself;    -   changes in their metabolic activities; and    -   changes in their local distribution.

The dysbiosis hypothesis states that the modern diet and lifestyle, andalso the use of antibiotics and various antimicrobials in theenvironment, have led to the disruption of the normal intestinalmicrobiota. These factors result in alterations in bacterial metabolism,as well as the overgrowth of potentially pathogenic microorganisms.Altered microbiota is now believed to play a role in several diseaseconditions, including disorders like irritable bowel syndrome (IBS),inflammatory bowel disease (MD), gout, pouchitis, chronic kidneydisease, psoriasis, frailty and various metabolic diseases includingobesity and type 2 diabetes.

Type 2 diabetes (T2D) is a metabolic disorder characterized byhyperglycemia and defects in insulin secretion and action. T2D is on therise worldwide and an estimated 350 million people will be affected by2030. This chronic disease is associated with multiple metabolic andcardiovascular comorbidities, and increased mortality fromcardiovascular complications. Equally alarming is the fact that abouthalf of all patients with T2D are newly detected, and many of them havecardiovascular complications at the time of diagnosis. Long beforediabetes develops, impaired glucose tolerance (IGT) and other metabolicdefects may appear. Since pharmacological and lifestyle interventionscan reduce or postpone diabetes, especially in subjects with IGT, earlydetection of individuals at risk of T2D is important for prevention andfor reducing the costs of medical care.

T2D is a result of complex gene-environment interactions, and severalrisk factors have been identified, including age, family history, diet,sedentary lifestyle, and obesity. The newly added factors associated tothe microbiota composition, including the presence of specific bacterialgenera and species in the gastro intestinal tract of a person can beused alone, or in combination with other measurements such as Body MassIndex (BMI), waist-to-hip ratio (WHR), waist circumference (WC) andspecific markers, to better predict whether an individual is at risk fordeveloping type 2 diabetes. Further can the information related tospecific changes of the microbiota composition be used to formulateintervention options to correct the specific dysbiosis using certainprobiotic products.

The gut microbiota has been proposed as an environmental factor thataffects body metabolism and insulin sensitivity and has also been foundto be altered in obesity. However, the relationship between the gutmicrobiota and T2D has not until recently been studied in large humancohorts, for example in the study by Karlsson et al. (Nature, Jun. 6,2013). In addition, some gut microbial markers have recently beenassociated to T2D, for example in the metagenomic study of Chinesediabetic patients published by Qin et al. (Nature, Sep. 26, 2012).

During pregnancy—usually around the 24th week—many women developgestational diabetes (GDM). History of previous gestational diabetes isknown to be a risk condition for later development of type 2 diabetes.High glycemic levels during pregnancy or after delivery, are strongpredictors of diabetes development in women. Relative hyperglycemiatypically indicates some degree of insulin resistance and beta-celldysfunction, which can be observed in such women, even with normal bodyweight and glucose tolerance.

Identifying and treating women with GDM is also important to minimizematernal and neonatal morbidity, preeclampsia and problems with highbirth weights. Many of the immune and metabolic changes occurring duringnormal pregnancy are similar to those also described in the metabolicsyndrome. The gut microbiota can, as described above, play a role invarious diseases associated with the metabolic syndrome. A study byKoren et al. (Cell. 2012 Aug. 3) showed that the intestinal microbiotachanges dramatically from first (T1) to third (T3) trimesters.

Inflammatory bowel disease (IBD) involves chronic inflammation of all orpart of the digestive tract. IBD primarily includes ulcerative colitisand Crohn's disease. Both usually involve severe diarrhea, pain, fatigueand weight loss. Abundant data have incriminated intestinal bacteria inthe initiation and amplification stages of IBD.

Gout is often characterized by recurrent outbreaks of inflammatoryarthritis with a red, tender, hot, and swollen joint. This is associatedwith a lot of pain which typically comes on rapidly. The underlyingmechanism involves elevated levels of uric acid in the blood anddiagnosis may be confirmed by seeing the crystals in joint fluid ortophus. The cause of gout is a combination of diet and genetic factors.In recent decades, gout has become more common and the increases inonset and recurrences of gout likely reflect changes in demographicfactors. Notable among these factors are increased longevity andage-associated cardiovascular, metabolic, and renal diseases in thepopulation; the use of medications that alter urate balance as anunintended consequence of treatment for these chronic disorders; andincreased dietary intake of foods and food additives that contribute tothe development of obesity and diabetes mellitus.

Pouchitis is an inflammation of the lining of a pouch that is surgicallycreated in the treatment of ulcerative colitis and certain otherdiseases. The pouch is attached internally to the area just above theanus to hold waste before it's eliminated. Pouchitis is the mostfrequently observed long-term complication of an ileal pouch-analanastomosis (IPAA). Symptoms can include diarrhea, abdominal pain andjoint pain, cramps, fever, increased number of bowel movements,nighttime fecal seepage, fecal incontinence, and a strong feeling of theneed to have a bowel movement. The majority of patients with acutepouchitis respond to initial therapy with antibiotics, but approximately60 percent have at least one recurrence.

Chronic kidney disease, sometimes also referred to as chronic kidneyfailure, is characterised by the gradual loss of kidney function and isfurther defined as the presence of kidney damage (usually detected asurinary albumin excretion of ≥30 mg/day, or equivalent) or decreasedkidney function (defined as estimated glomerular filtration rate[eGFR]<60 mL/min/1.73 m2) for three or more months, irrespective of thecause. When chronic kidney disease reaches an advanced stage, dangerouslevels of fluid, electrolytes and wastes can build up in the body.Available treatment for chronic kidney disease does focus on slowing theprogression of the kidney damage. Chronic kidney disease can progress toend-stage kidney failure, which is fatal without artificial filtering(dialysis) or a kidney transplant.

Psoriasis is a common chronic skin disorder which is characterized bypatches of abnormal skin. These patches can be red, itchy and scaly.Psoriasis is generally seen as a genetic disease which is triggered byenvironmental factors. No cure is available for the disease but thereare various treatments that can help to control and balance thesymptoms.

Frailty is a common geriatric syndrome that embodies an elevated risk ofcatastrophic declines in health and function among older adults. Thereis no gold standard for diagnosing frailty, however frail patients oftenpresent an increased burden of symptoms and medical complexity andreduced tolerance for medical interventions.

The connection between gut microbiota and energy homeostasis andinflammation and its role in the pathogenesis of obesity areincreasingly recognized. Animal models as well as human data connect analtered microbiota composition to the development of obesity in the hostthrough several mechanisms.

The bacterial species Faecalibacterium prausnitzii is one of the mostabundant bacteria in the human gut ecosystem and it is an importantsupplier of various metabolites to the intestinal epithelium. Low ordepleted numbers of Faecalibacterium have been associated with forexample IBD, gout, pouchitis, chronic kidney disease, psoriasis, frailtyand also T2D and GDM. Some have suggested the use of certain sources ofsubstrates that promote growth of beneficial bacteria, such as forexample carbon, so called prebiotics, including inulin, as a way toincrease the biomass of for example Faecalibacterium. One potentialproblem with that approach is that firstly the specific strains ofFaecalibacterium must be present in the gastro intestinal tract,secondly the prebiotic must reach the location where the targetedFaecalibacterium strains are located and thirdly, and importantly, theprebiotic may also “feed” other bacteria, which may be highly unwanted,especially when a targeted intervention is needed.

A preferred approach for intervention would be to give a probioticproduct containing the wanted strains and preferably also in such way topromote their activity and increase their biomass in the proper locationof the gastrointestinal tract. However bacteria such as Faecalibacteriumare very difficult to colonize after delivery to the human intestine.Another hurdle when isolating and culturing representatives of gutmicrobiota for developing new potential probiotics is the complexity ofgut environment in terms of distinct niches and habitats. Also, severalgut microbes are cross fed from other microbes and may also require somegrowth factors from the host. The present invention is intended to solvethis problem.

Definitions

All terms used in the present specification are intended to have themeaning usually given to them in the art. For the sake of clarity, someterms are also defined below.

Throughout the text, the term “Type 2 diabetes” (T2D) is used to referto a metabolic disorder characterized by hyperglycemia, insulinresistance and relative impairment in insulin secretion.

The term “metagenomics” refers to the application of modern genomicstechniques to the study of communities of microbial organisms directlyin their natural environments, bypassing the need for isolation and labcultivation of individual species.

“Probiotics” are live microorganisms that, when administered in adequateamounts, confer a health benefit to the host.

“Synergy” is the interaction of two or more agents (for example two ormore different microorganisms), entities, factors or substances so thattheir combined effect, “the synergistic effect” is greater than the sumof their individual effects.

“Symbiosis” is a close, prolonged association between two or moredifferent organisms of different species that may, but does notnecessarily, benefit each member.

SUMMARY OF THE INVENTION

The invention herein relates to methods and products for probioticinterventions in mammals based on certain anaerobic bacteria.

A primary object of the invention is to support the growth andcolonization in humans of a strain of the species Faecalibacteriumprausnitzii by utilizing a unique electron cross talk and symbiosisbetween the bacterial species Faecalibacterium prausnitzii andDesulfovibrio piger (or alternative strain as defined elsewhere herein).The symbiosis can lead to the synergistic effect of more butyrateproduction than F. prausnitzii alone, as well as resulting in anincrease in biomass or numbers of Faecalibacterium prausnitzii in thegastrointestinal tract. Thus, the present invention provides methods forthe increasing the growth and colonization (e.g. in the gastrointestinaltract, preferably in humans) of Faecalibacterium prausnitzii bacteria.

Another primary object of the invention is to use the symbiotic strainsfor treatment and or prevention of diseases associated with reducedbutyrate production or diseases associated with reduced or low levels ofFaecalibacterium prausnitzii. Thus, in one embodiment the presentinvention provides a strain of Faecalibacterium prausnitzii (F.prausnitzii) and/or a strain of Desulfovibrio piger (D. piger) for usein therapy, e.g. as probiotics.

Another object of the invention is to use a strain of D. piger (oralternative strain as described herein) for the treatment and/orprevention of diseases associated with reduced butyrate production ordiseases associated with reduced or low levels of Faecalibacteriumprausnitzii. D. piger will boost endogenous F. prausnitzii.

Any appropriate strain of Faecalibacterium prausnitzii can be used inthe present invention. Faecalibacterium prausnitzii is an anaerobicbacteria and in particular, appropriate strains of Faecalibacteriumprausnitzii will have (i) the ability to produce butyrate. In addition,appropriate strains of Faecalibacterium prausnitzii will have one ormore (e.g. 1 or 2), or preferably all, of the ability to (ii) consumeacetate, (iii) produce extracellular electrons and (iv) to producelactate. In some embodiments, strains with features (i), (ii) and (iv)are used. Strains with the ability to produce lactate (in particularhigh or significant levels of lactate) are particularly preferred.Typically these Faecalibacterium prausnitzii strains are glucosefermenting which results for example in the conversion of glucose tolactate and butyrate.

Even though any appropriate strain of Faecalibacterium prausnitzii canbe used in the present invention the inventors have identified someunique functions when comparing different strains of Faecalibacteriumprausnitzii. Strains with the presence of the enzyme L-lactatedehydrogenase (or strains containing an L-lactate dehydrogenase genewhich in turn results in production or expression of the active enzyme)provides the bacteria with better properties to produce lactate and arepreferred as this is more beneficial for the D. Piger strain in theirsynergistic relationship. Exemplary L-lactate dehydrogenase genes orenzymes are defined as EC 1.1.1.27 genes/enzymes.

A particularly preferred strain of Faecalibacterium prausnitzii for usein the present invention is denoted herein as Faecalibacteriumprausnitzii strain FBT-22 and has been deposited under the BudapestTreaty at DSMZ (Leibniz Institute DSMZ—German Collection ofMicroorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig,Germany) on Oct. 20, 2015 and has been given the accession number DSM32186. This particular strain has L-lactate dehydrogenase and showshigher lactate production compared to other strains of Faecalibacteriumprausnitzii. More lactate during growth provides more substrate for D.piger and therefore supports their interaction.

This strain, e.g. the isolated strain, and its use in therapy, e.g. as aprobiotic, e.g. for the treatment of diseases as described elsewhereherein, provides a yet further aspect of the invention.

Another preferred strain of Faecalibacterium prausnitzii for use in thepresent invention is the strain denoted as A2-165, which is availablefrom the DSMZ (Leibniz Institute DSMZ—German Collection ofMicroorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig,Germany) and has the accession number DSM 17677.

Similarly, for the Desulfovibrio piger strain component, it is envisagedthat any appropriate strain of Desulfovibrio piger may be used.Desulfovibrio piger is an anaerobic bacteria and in particular, anappropriate Desulfovibrio piger strain will have one or more (e.g. 1 or2), or preferably all, of the characteristics of being (i) acetateproducing, (ii) lactate consuming and (iii) having the ability to be anelectron acceptor (e.g. to reduce more sulfate to sulfide). In someembodiments, strains with features (i) and (ii) are used. Such strainscan generally convert lactate to acetate. As these particularcharacteristics of Desulfovibrio piger bacteria are important for thesymbiosis with the Faecalibacterium prausnitzii bacteria, it isenvisaged that any other species of bacteria (i.e. alternative strains)which have these characteristics can also be used in the presentinvention. A particularly preferred strain of Desulfovibrio piger foruse in the present invention is denoted herein as Desulfovibrio pigerstrain FBT-23 and has been deposited under the Budapest Treaty at DSMZ(Leibniz Institute DSMZ—German Collection of Microorganisms and CellCultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on Oct. 20,2015 and has been given the accession number DSM 32187.

This strain, e.g. the isolated strain, and its use in therapy, e.g. as aprobiotic, e.g. for the treatment of diseases as described elsewhereherein, provides a yet further aspect of the invention.

Preferably the Faecalibacterium prausnitzii and Desulfovibrio piger (oralternative) strains are used together as a combination therapy.

Preferably the strains for use in the invention are isolated strains,e.g. strains which are isolated from the human or animal body. Suchstrains are thus separate from other strains of microorganisms or otherimpurities from the human or animal body from which they are isolated,e.g. are pure cultures, except when two or more isolated strains aremixed or combined together for use in the present invention.

When the two strains interact (e.g. the Faecalibacterium prausnitzii andDesulfovibrio piger strains described above), for example areco-cultivated (co-cultured) or administered together, or otherwise comeinto contact with each other, then butyrate production is increased whenboth strains are present compared to the butyrate production by theFaecalibacterium prausnitzii strain alone. Preferably the effect on theincrease in butyrate production is a synergistic effect. Wherenecessary, conditions or the environment in which the interaction orcontact between the two strains used in the invention takes place can beselected to support butyrate production.

Thus, a yet further embodiment of the invention provides a compositionor product comprising the Faecalibacterium prausnitzii and Desulfovibriopiger (or alternative) strains as defined herein either individually orin combination. Preferred combinations of bacteria are those that giverise to a synergistic effect on butyrate production when the strainsinteract with each other. Such interaction is a form of symbiosis.

As outlined above, the strains and combinations thereof as describedherein have therapeutic utility, and can be used in the treatment orprevention of any disease which will benefit from increased productionof butyrate or any disease which will benefit from an increase in numberor colonization of Faecalibacterium prausnitzii bacteria. Appropriatediseases thus include any disease associated with reduced butyratelevels, e.g. reduced production of butyrate (or a depletion of butyrateor a lack of butyrate) caused for example by a reduction or depletion inmicroorganisms which produce butyrate, for example a disease associatedwith reduced or low number or depleted amounts of Faecalibacteriumprausnitzii bacteria, or caused for example by a reduction in butyrateproduction by microorganisms which produce butyrate. Alternatively, thestrains and combinations thereof as described herein can be used in thetreatment or prevention of any disease associated with a reduction ordepletion in microorganisms which produce butyrate, for example adisease associated with reduced or low number or depleted amounts ofFaecalibacterium prausnitzii bacteria. These diseases are typicallydiseases of the gastrointestinal (GI) tract.

The reference to reduced levels, reduced production, or depletion or lowlevels, or low numbers, etc., will be readily understood and determinedby a skilled person, for example by comparison to the levels found in anappropriate control, e.g. in a healthy patient. Thus, such levels ornumbers etc., may be regarded as below normal, or sub-normal orabnormal, or otherwise less abundant than normal. Preferably suchreductions (and indeed other reductions or decreases or negative effectsas mentioned elsewhere herein) are measurable reductions, morepreferably they are significant reductions, preferably clinicallysignificant or statistically significant reductions, for example with aprobability value of ≤0.05, when compared to an appropriate controllevel or value.

Indeed, where significant changes are described herein, it is preferredthat such changes are statistically significant changes, for examplewith a probability value of ≤0.05, when compared to an appropriatecontrol level or value.

Exemplary diseases are described elsewhere herein and include T2D, GDM,obesity, gout, pouchitis, chronic kidney disease, psoriasis, frailty,IBD, IBS, abdominal pain associated with IBS and constipation (ordiseases associated with constipation). Preferred diseases to be treatedare T2D or GDM. Another preferred disease or condition to be treated isdysbiosis.

Thus, viewed alternatively, the present invention provides therapeuticmethods for the treatment or prevention of T2D, GDM, obesity, gout,pouchitis, chronic kidney disease, psoriasis, frailty, IBD, IBS,abdominal pain associated with IBS or constipation (or diseasesassociated with constipation) or other diseases or conditions asdescribed herein (e.g. dysbiosis), or for the treatment or prevention ofmetabolic diseases, comprising the administration of probiotic strainsas described herein.

In all embodiments described herein the term “disease associated with”can also refer to “disease characterised by”.

The symbiotic and preferably synergistic effects of the two strains oneach other result in the increased production of butyrate and in theincreased growth and colonization of Faecalibacterium prausnitziibacteria in the GI tract thereby alleviating and treating disease.Preferably, the strains are selected such that the total butyrateproduction by both strains when present together (e.g. in a co-cultureor when administered together) is greater than the butyrate productionobserved with the Faecalibacterium prausnitzii bacteria alone and ispreferably synergistic, i.e. the total butyrate production by bothstrains is greater (increased), preferably significantly greater(increased), than the sum of the individual levels of butyrateproduction.

The present invention thus further provides a strain of Faecalibacteriumprausnitzii as described herein and a strain of Desulfovibrio piger (oralternative strain) as described herein for use in therapy by combined,sequential or separate administration.

Further provided is a product or composition comprising a strain ofFaecalibacterium prausnitzii and a strain of Desulfovibrio piger (oralternative strain) as described herein as a combined preparation forseparate, simultaneous or sequential use in the treatment or preventionof diseases as defined elsewhere herein.

Thus, the present invention provides a strain of Faecalibacteriumprausnitzii (F. prausnitzii) and a strain of Desulfovibrio piger (D.piger) (or alternative strain) as described herein for use in thetreatment of diseases as described herein, e.g. a disease associatedwith reduced levels or production of butyrate in the GI tract (forexample due to a reduction or depletion in microorganisms which producebutyrate, for example a disease associated with reduced or low number ordepleted amounts of Faecalibacterium prausnitzii bacteria, or areduction in butyrate production by microorganisms which producebutyrate), or other diseases as described elsewhere herein.

Viewed alternatively, the present invention provides a strain ofFaecalibacterium prausnitzii (F. prausnitzii) and a strain ofDesulfovibrio piger (D. piger) (or alternative strain) as describedherein for use in the manufacture of a medicament or composition for thetreatment of diseases as described herein, e.g. a disease associatedwith reduced levels or production of butyrate in the GI tract (forexample due to a reduction or depletion in microorganisms which producebutyrate, for example a disease associated with reduced or low number ordepleted amounts of Faecalibacterium prausnitzii bacteria, or areduction in butyrate production by microorganisms which producebutyrate), or other diseases as described elsewhere herein.

In an alternative aspect, the present invention provides the use of theproducts, strains or compositions as described herein in the manufactureof a medicament or composition for use in the treatment or prevention ofdiseases as described herein.

Viewed alternatively, the present invention provides a method oftreating a disease as described herein, e.g. a disease associated withreduced levels or production of butyrate in the GI tract (for exampledue to a reduction or depletion in microorganisms which producebutyrate, for example a disease associated with reduced or low number ordepleted amounts of Faecalibacterium prausnitzii bacteria, or areduction in butyrate production by microorganisms which producebutyrate), or other diseases as described elsewhere herein, in apatient, said method comprising administration of an effective amount ofa strain of Faecalibacterium prausnitzii (F. prausnitzii) and a strainof Desulfovibrio piger (D. piger) (or alternative strain) as describedherein to said patient.

The administration of the probiotic strains in said methods of treatmentand uses of the invention (or in any other methods of treatment ortherapeutic uses as described herein) is carried out in pharmaceuticallyor physiologically effective amounts, to subjects in need of treatment.Thus, said methods and uses may involve the additional step ofidentifying a subject in need of treatment.

In all the embodiments described herein, preferred diseases to betreated are T2D or GDM. Preferred strains for use in therapy or in thetreatment of the diseases described herein, and in particular T2D orGDM, are the Faecalibacterium prausnitzii strain DSM 32186 or theDesulfovibrio piger strain DSM 32187, which can also be used incombination. Another preferred Faecalibacterium prausnitzii strain isA2-165 (DSM 17677).

As set out above, a yet further aspect of the invention provides astrain of D. piger (or alternative strain as described herein) for usein the treatment or prevention of a disease associated with reducedbutyrate levels or a disease associated with reduced or low numbers ofFaecalibacterium prausnitzii bacteria.

Thus, the present invention also provides a strain of D. piger (oralternative strain as described herein) for use in the manufacture of amedicament or composition for the treatment of diseases as describedherein.

The present invention also provides a method of treating a disease asdescribed herein, in a patient, said method comprising administration ofan effective amount of a strain of Desulfovibrio piger (or alternativestrain as described herein) to said patient.

Preferred embodiments for said methods and uses, e.g. preferred strainsof Desulfovibrio piger (or alternative strains) and preferred diseasesare as described elsewhere herein. In such embodiments theadministration of D. piger will boost or increase (e.g. increase thebiomass or numbers of, or otherwise increase the growth or colonizationof, as described elsewhere herein) the endogenous F. prausnitzii in thepatient or subject, e.g. in the gastrointestinal tract of the patient orsubject.

Other objects and advantages of the present invention will becomeobvious to the reader and it is intended that these objects andadvantages are within the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the major fermentative pathway employed by gut microbiotain human gut.

FIG. 2 shows the symbiosis between F. prausnitzii and Desulfovibriopiger.

FIG. 3 shows the comparative SCFA profiles of F. prausnitzii isolateFBT-22 and type strain A2-165 in physiologically relevant growth medium,Brain Heart Infusion (LYBHI), y axis in mM.

FIG. 4 shows the metabolic profiles of F. prausnitzii type strain A2-165monoculture and coculture with D. piger in LYBHI growth medium, y axisin mM.

FIG. 5 shows the metabolic profiles of F. prausnitzii isolate FBT-22monoculture and coculture with D. piger in LYBHI growth medium, y axisin mM.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

As set out above, the invention herein relates to methods and productsfor probiotic interventions in mammals based on certain anaerobicbacteria.

It can be seen that in preferred embodiments of the invention, acombination or mixture of probiotic bacteria are administered(combination therapy). Such mixtures or combinations of probioticbacteria can be administered together in a single (the same) compositionor administered separately (e.g. in different products or compositions).If administered separately then such administration may be sequential orsimultaneous. However, the separate administration forms part of thesame therapeutic regimen or method.

In embodiments where the administration of the two strains is separateor sequential, it is preferred that the administrations are made withina reasonable time frame of each other. For example, the separateadministrations are preferably made within hours (e.g. one hour) orminutes (e.g. within 15 or 30 minutes) of each other, most preferablywithin as short a timeframe as possible (including simultaneous oreffectively simultaneous administration). Preferably the two strains ofprobiotic bacteria are co-administered in a single composition.

In embodiments where more than one strain of probiotic bacteria is usedin a mixture in a single composition, or where more than one strain ofprobiotic bacteria is used but they are administered separately, thenany appropriate ratio of the bacteria can be used providing that theprobiotic function of the strains (for example increased butyrateproduction and preferably a synergistic effect on butyrate production)is retained to a useful extent. Such ratios can readily be determined bya person skilled in the art. For example, such a combination of twostrains (e.g. F. prausnitzii: D. piger (or alternative strain)) might beused at a ratio of 1:10, 1:5, 1:1, 5:1, or 10:1 or anywhere betweenthese extremes, e.g. 1:1. Such ratios may also be used in the products,kits, compositions, etc., of the invention as described elsewhereherein.

The present invention thus provides combination therapies using at leasttwo strains of probiotic bacteria, preferably in which the strains havea symbiotic and more preferably a synergistic effect on each other, inparticular in terms of increased butyrate production.

Such synergistic effects (or indeed increased effects) can readily bemeasured in vitro in order to select appropriate combinations ofbacteria, for example by measuring the levels of butyrate productionfrom each individual strain alone by any appropriate assay and thenassessing whether the butyrate production of the strains in combination(e.g. when co-cultured or otherwise able to interact with each other) isgreater, and preferably significantly greater, than the sum of the levelof butyrate production by the individual strains. A simple increase(preferably a significant increase) in butyrate production can also bemeasured in this way. Appropriate assays may be carried out underanaerobic conditions. Appropriate exemplary methods are described in theExamples.

In the therapeutic methods and uses as described herein, the strains areadministered in appropriate doses, formulations etc. such that butyrateproduction is increased in the GI tract of the mammal. In addition,preferably the numbers of Faecalibacterium prausnitzii bacteria areincreased (e.g. the biomass of Faecalibacterium prausnitzii isincreased) or growth and colonization of the Faecalibacteriumprausnitzii bacteria within the GI tract is improved.

Preferably such increases (and indeed other increases, improvements orpositive effects as mentioned elsewhere herein) are measurableincreases, etc., (as appropriate), more preferably they are significantincreases, preferably clinically significant or statisticallysignificant increases, for example with a probability value of ≤0.05,when compared to an appropriate control level or value (e.g. compared toan untreated or placebo treated subject or compared to a healthy ornormal subject, or the same subject before treatment).

The methods and uses of the present invention are suitable for theprevention of diseases as well as the treatment of diseases. Thus,prophylactic treatment is also encompassed by the invention. For thisreason in the methods and uses of the present invention, treatment alsoincludes prophylaxis or prevention where appropriate.

The methods and uses of the invention can be carried out on any mammal(patient or subject), for example humans or any livestock, domestic orlaboratory animal. Specific examples include mice, rats, pigs, cats,dogs, sheep, rabbits, cows and monkey. Preferably, however, the mammalis a human.

In a further aspect of the invention kits or pharmaceutical packs areprovided. Thus, the present invention provides a kit or pharmaceuticalpack comprising (or consisting of):

(i) a strain of Faecalibacterium prausnitzii as defined herein, and

(ii) a strain of Desulfovibrio piger (or alternative strain) as definedherein; or a kit or pharmaceutical pack comprising (or consisting of):

a strain of Desulfovibrio piger (or alternative strain) as definedherein, for example to boost growth of endogenous F. prausnitzii asdescribed elsewhere herein.

As described elsewhere herein, the two strains forming separatecomponents of the kit can be administered as separate components or canbe combined together before administration. In alternative embodimentsof the kit, the two strains could be provided together as a single kitor pack component. Preferably such kits or packs are for use in themethods and uses of the present invention, for example for use in thetreatment of diseases as defined herein. The kit or pack optionally alsocomprises instructions for administration of the components, orinstructions for use of the kit or pack.

Earlier studies are indicating that the bacterial alterations observedin Type 2 diabetes (T2D) do not affect the overall composition of themicrobiota but the abundance of a limited number of species.Specifically there is a reduction of bacteria producing the short chainfatty acid butyrate and that depletion of butyrate producing bacteriaalso reduces the levels of secondary bile acids. This seems to be truealso in gestational diabetes (GDM) disease and other metabolicassociated diseases. Faecalibacterium, which is a butyrate-producer withanti-inflammatory effects, also depleted in inflammatory bowel disease,has been reported to be less abundant on average in pregnant women withGDM at the third trimester (T3).

Based on our own studies in healthy humans and humans with disease usingmetagenome analysis we isolated several strains of Faecalibacteriumprausnitzii from human faeces and selected the most promising ones forfurther development as potential probiotic strains. Based on thesestudies, appropriate strains of Faecalibacterium prausnitzii for use inthe present invention are described elsewhere herein. A preferred strainis FBT-22 (DSM 32186). This particular strain has the L-lactatedehydrogenase gene and shows higher lactate production compared to otherstrains of Faecalibacterium prausnitzii. Indeed, as mentioned elsewhereherein, strains with the ability to produce lactate (in particular highor significant levels of lactate) are particularly preferred. Exemplaryappropriate levels of lactate production by the strains for use in theinvention are those which are sufficient to have a biological effect,for example a positive effect on the growth of D. piger upon co-culture.Exemplary levels are levels of at least 10 mM, 20 mM, 30 mM, 40 mM, 50mM, or 60 mM, e.g. when strains are cultured in an appropriate growthmedium, e.g. PGM or LYBHI as described in the Examples. Exemplary highlevels of lactate production are levels of at least 35 mM, 40 mM, 45 mM,50 mM, 55 mM or 60 mM, e.g. when strains are cultured in an appropriategrowth medium, e.g. PGM or LYBHI as described in the Examples. Strainswhich contain a L-lactate dehydrogenase gene (or express an activeL-lactate dehydrogenase enzyme) are particularly preferred.

As mentioned elsewhere herein, Faecalibacterium prausnitzii strains withthe ability to produce butyrate are particularly preferred. Exemplaryappropriate levels of butyrate production by the strains for use in theinvention are those which are sufficient to have a biological effect,for example a positive effect on T2D or other diseases as describedherein. Exemplary levels are levels of at least 3 mM, 5 mM, 7 mM, 10 mM,or 15 mM, or up to 10 mM or 15 mM, e.g. when strains are cultured in anappropriate growth medium, e.g. PGM or LYBHI as described in theExamples. Preferably, in accordance with the present invention, thelevel of butyrate production is increased, preferably significantlyincreased, when the strain of Faecalibacterium prausnitzii is present incombination with a strain of D. piger (or alternative strains) comparedto the butyrate production by Faecalibacterium prausnitzii alone.Preferably a synergistic increase is observed. Exemplary levels ofincrease are at least 1.5 fold, 2.0 fold, 2.5 fold, 3.0 fold or 3.5fold.

F. prausnitzii is the most abundant bacterium in the human intestinalmicrobiota of healthy adults, representing up to more than 5% of thetotal bacterial population. Over the past five years, an increasingnumber of studies have clearly described the importance of this highlymetabolically active commensal bacterium as a component of the healthyhuman microbiota. Changes in the abundance of F. prausnitzii have beenlinked to dysbiosis in several human disorders.

F. prausnitzii is an extremely oxygen sensitive (EOS) bacterium and isdifficult to cultivate even in anaerobic conditions. The major endproducts of glucose fermentation by F. prausnitzii strains are formate,lactate and substantial quantities of butyrate (>10 mM butyrate invitro).

Some observations provide key insight in host-microbe interactions atthe gut barrier of F. prausnitzii. The strain is:

-   -   Butyrate producing    -   Glucose fermenting    -   Acetate consuming    -   Able to produce extracellular electrons, e.g. capable to respire        electrons to extracellular electron acceptors via riboflavin.    -   Using glucose as electron donor and flavin as mediator.

Considering the abundance and growth of F. prausnitzii in a healthy gutour assumption was that there must be other bacteria in the vicinitythat can act in a symbiotic fashion and support the growth of F.prausnitzii. So by the identified characteristics we looked for abacteria that is lactate consuming, acetate producing and also capableof acting as an electron acceptor. Based on these characteristics astrain can be chosen that together with F. prausnitzii act in asymbiotic fashion. One possible candidate was found in Desulfovibriopiger (and a particularly preferred strain of Desulfovibrio piger isFBT-23 (DSM 32187). However, any other strains with these properties(i.e. alternative strains) could also be used.

Desulfovibrio is one of the first genera described and probably the mostthoroughly studied genus among the sulfate-reducing bacteria. They aresulfate-reducing, nonfermenting, anaerobic, gram-negative bacillicharacterized by the presence of a pigment, desulfoviridin, whichfluoresces red in alkaline pH and blue-green in acid pH underlong-wavelength UV light. D. piger strains have never been isolated fromoutside the human body and can be considered natural inhabitants of theintestinal tract, where sulfates abound.

Some characteristics of the strain are the following:

-   -   Converts lactate or consumes lactate, a reduced fermentative        metabolite of microbes, into the less reduced product acetate.    -   The conversion of lactate to acetate releases electrons, which        reduces sulfate to sulfide.    -   Desulfovibrio cytochromes may act as electron acceptors from the        surrounding reduced environment or bacteria and are utilized to        reduce sulfate to sulfide.

To our surprise when a F. prausnitzii bacterial cell is in the vicinityof a D. piger bacterial cell there is a unique electron cross talk andsymbiosis between F. prausnitzii and D. piger. This new discovery isutilised by the invention herein by making products based on thecombination of selected strains of the two species to support the growthand colonization in humans of a strain of F. prausnitzii. Anotherpossible product would be to use D. piger alone to administer, e.g. tohumans, to support growth of endogenous F. prausnitzii.

The symbiosis between the two species can in part be described by thefollowing, see FIG. 2:

-   -   F. prausnitzii converts glucose to lactate and butyrate, consume        acetate and produces extracellular electrons    -   D. piger converts lactate to acetate and accepts electrons to        reduce more sulfate to sulfide.    -   Net results are better growth of both organisms and more        butyrate production, showing a synergistic effect.    -   This interaction has been confirmed by showing higher butyrate        accumulations in cocultures of F. prausnitzii and D. piger (see        Example 4).

The symbiosis results in the wanted growth and multiplied production ofbutyrate by F. prausnitzii (see Example 4) to be used, according to theinvention herein, for probiotic intervention in people at risk, orhaving, dysbiosis in their intestinal microbiota (e.g. gastrointestinalmicrobiota dysbiosis) where butyrate producing bacteria are reduced suchas in T2D or GDM.

Appropriate probiotic strains for use in the invention can be obtainedor isolated from any mammal which is capable of suffering from or issusceptible to gastrointestinal microbiota dysbiosis especially in T2D,GDM obesity, gout, pouchitis, chronic kidney disease, psoriasis,frailty, IBD, IBS, abdominal pain associated with IBS and constipationdiseases, or other diseases as described herein. Humans are a preferredsource. Appropriate sample types from which to obtain appropriateprobiotic bacteria would be well known to a person skilled in the art.However fecal samples are preferred. Appropriate culture conditions areanaerobic conditions. Appropriate culture medium can be selected by aperson skilled in the art, for example PGM medium or LYBHI or othermedium as described in the Examples.

It is a further object of the invention to provide methods, kits,systems, compositions and products for said symbiosis.

Thus, the strains (or combinations thereof) as described herein may takethe form of a compound (agent) or composition, e.g. a pharmaceuticalcompound or composition or a nutritional compound or composition.

The present invention thus also provides a composition or formulationcomprising a F. prausnitzii strain as described herein and a D. pigerstrain or an alternative bacterial strain as described herein, e.g.which has one or more of the characteristics of: (i) being acetateproducing, (ii) being lactate consuming and (iii) having the ability tobe an electron acceptor; and at least one additional component selectedfrom the group consisting of a carrier, diluent or excipient (forexample a pharmaceutically acceptable carrier, diluent or excipient), afoodstuff or food supplement, or a further therapeutic or nutritionalagent. Thus, said compositions can be formulated as pharmaceuticalcompositions or as nutritional compositions, e.g. as a food product.

Therapeutic uses of the strains, compositions and formulations of theinvention as defined herein, are also provided.

An appropriate mode of administration and formulation of the strains,compositions, formulations, etc., is chosen depending on the site ofdisease. A preferred mode of administration is oral or rectal, however,equally intravenous or intramuscular injection may be appropriate.

Appropriate doses of the strains, compositions and formulations of theinvention as defined herein can readily be chosen or determined by askilled person depending on the disorder to be treated, the mode ofadministration and the formulation concerned. For example, a dosage andadministration regime is chosen such that the probiotic bacteria (orcombination of bacteria) administered to the subject in accordance withthe present invention can result in a therapeutic or health benefit(e.g. an increase in the butyrate levels in the gastrointestinal tractor an increase in the growth of F. prausnitzii bacteria in thegastrointestinal tract or the treatment of disease). Thus, inembodiments of the invention where two different strains of bacteria areadministered, an appropriate dose of each bacteria is selected such thata therapeutic or health benefit is observed when both strains arepresent. For example, daily doses of one or each bacteria of 10⁴ to10¹², for example 10⁵ to 10¹⁰, or 10⁶ to 10⁸, or 10⁸ to 10¹⁰ total CFUsof bacteria may be used. A preferred daily dose of one or each bacteriais around 10⁸ or 10⁹ total CFUs, e.g. 10⁷ to 10¹⁰ or 10⁸ to 10¹⁰ or 10⁸to 10⁹.

Thus, products or compositions or formulations or kits containingstrains as defined herein are provided. Preferred products orcompositions comprise frozen, freeze-dried, lyophilized, or driedbacteria and are preferably in a unit-dosage format, e.g. a capsule ortablet or gel. Preferred products or compositions will contain both a F.prausnitzii strain and a D. piger (or alternative) strain. Appropriateratios and doses (e.g. in the form of numbers of bacteria or CFUs) foruse in such products, etc., are described elsewhere herein and in theExamples. Other components may also be included in such products, etc.,for example preservatives (e.g. glycerol), stabilizers, gelling agentsand/or cryoprotectants. In some embodiments such additional componentsare non-natural agents.

As shown in FIG. 1, theoretically homoacetate, homoformate and lactateproduction are the most energy efficient pathways in terms of electrondisposal when glucose was used as electron donor. Homobutyrogenesisproduces 4 moles of excess electrons per mole of glucose while lactateto acetate conversion yields 4 moles of electrons per mole of acetateproduction. Production of proprionate from glucose yields excess of 10moles of electrons. During fermentation, this electron disproportion isbalanced by mixed acid fermentation and gas productions. Some microbesrespire electrons to extracellular electron acceptors such as nitrate,sulfate or insoluble metallic complex. Moreover, oxygen can acts asindirect electron acceptor as some of the gut microbes can consumeoxygen in small amounts. The net result of microbial activitiesgenerates electron rich reducing environment in the human or animal gut,which keeps the net redox potential negative. This oxygen deficientreducing environment favours the growth of strict anaerobes in the gutlumen. Despite the fact that there is continuous influx of oxygen viaingestion of food and diffusion from gut mucosa the net redox potentialof the gut remains negative, ca −300 mV.

The therapeutic uses of the invention as defined herein include thereduction, prevention or alleviation of the relevant disorder orsymptoms of disorder (e.g. can result in the modulation of diseasesymptoms). Such relevant disorders are described elsewhere herein, forexample those associated with lack of or depleted butyrate production orthose associated with reduced or low numbers of F. prausnitzii, as forexample in T2D, GDM, obesity, gout, pouchitis, chronic kidney disease,psoriasis, frailty, IBD, IBS, abdominal pain associated with IBS, andconstipation diseases. The reduction, prevention or alleviation of adisorder or symptoms thereof can be measured by any appropriate assay.Preferably the reduction or alleviation of a disorder or symptoms isclinically and/or statistically significant, preferably with aprobability value of <0.05. Such reduction, prevention, or alleviationof a disorder or symptoms are generally determined compared to anappropriate control subject or population, for example a healthy subjector an untreated or placebo treated subject, or the baseline level in anindividual subject before treatment.

An appropriate mode of administration and formulation of the therapeuticagent is chosen depending on the treatment. A preferred mode ofadministration for probiotic bacteria or other supplements is oral orrectal.

Symbiotic combinations, or indeed any combinations of probiotic bacteriaas described herein, can be prepared in oil based liquids (for example:medium chain triglycerides) using lyophilized material.

The preferred viability of the each strain in raw material ranges from10⁶ CFU/g to 10¹⁰ CFU/g.

Administration into the subjects can be achieved via any of the deliverymode including enteric capsules, delayed or controlled release capsules,soft-gel capsules (e.g. chewable capsules), enteric coated capsules orcapsules within capsules.

Administration can be achieved via ALU-ALU based sachet form of dosagewith desiccant coatings.

Appropriate doses of the therapeutic agents as defined herein can bechosen by standard methods depending on the particular agent, the age,weight and condition of the patient, the mode of administration and theformulation concerned.

The therapeutic and prevention methods of the invention as describedherein can be carried out on any type of subject or mammal which iscapable of suffering from dysbiosis, e.g. gastrointestinal microbiotadysbiosis, especially in T2D, GDM obesity, gout, pouchitis, chronickidney disease, psoriasis, frailty, IBD, IBS, abdominal pain associatedwith IBS and constipation diseases. The methods are generally carriedout on humans.

DEPOSIT INFORMATION

A deposit of the proprietary bacterial strains Desulfovibrio piger DSM32187 and Faecalibacterium prausnitzii DSM 32186 have been made with theLeibniz Institute DSMZ (DSMZ)—German Collection of Microorganisms andCell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany. The dateof deposit for Desulfovibrio piger DSM 32187 and for Faecalibacteriumprausnitzii DSM 32186 was Oct. 20, 2015. Upon issuance of a patent, allrestrictions upon the deposits will be removed, and the deposits areintended to meet all of the requirements of 37 C.F.R. § 1.801-1.809. TheDSMZ has issued accession number DSM 32187 for Desulfovibrio piger DSM32187 and has issued accession number DSM 32186 for Faecalibacteriumprausnitzii DSM 32186. These deposits will be maintained in thedepository for a period of 30 years, or 5 years after the last request,or for the effective life of the patent, whichever is longer, and willbe replaced as necessary during that period. Applicants do not waive anyinfringement of their rights granted under this patent or under thePlant Variety Protection Act (7 U.S.C. 2321 et seq.).

The following are some examples of the invention, which are not meant tobe limiting of the use of the invention herein but to show practicalexamples in detail of how the invention may be used.

Example 1

Isolation of Faecalibacterium prausnitzii and Desulfovibrio piger asCo-Culture

Surprisingly, Faecalibacterium prausnitzii FBT-22 (DSM 32186) andDesulfovibrio piger strain FBT-23 (DSM 32187) were isolated from thefeces of healthy volunteer by microbiological pure culture techniqueunder strict anaerobic condition (5% H₂, 15% CO₂ and 80% N₂) employed ina Coy chamber. The Postgate medium (PGM) was employed as routine culturemedium for isolation and cultivation. The PGM contains (g/L);dipotassium phosphate: 0.5 g; ammonium chloride: 1; sodium lactate: 3.5;yeast extract: 1; Ascorbate: 0.1; cysteine: 0.5; sodium chloride: 1;peptone: 10; sodium sulphate: 1; calcium chloride dehydrate: 1;magnesium sulphate: 2; ferrous sulphate heptahydrate: 0.5. Sodiumsulphate, magnesium sulphate heptahydrate, calcium chloride dehydratewere autoclaved separately while ferrous sulfateheptahydrate was filtersterilized 0.22 μm filter and added after autoclaving and mixing of allcomponents. The final pH of the medium was adjusted with 1N NaOH or 1NHCl to 7.2±0.2.

The media was autoclaved at 100 kPa at 121° C. for 15 mins.

This medium lacks glucose which is the primary requirement for growth ofF. prausnitzii however, contains relatively high amounts of lactate. Therepeated subculturing leads to isolation of D. piger and F. prausnitzii.

Example 2

Alternative Isolation of a Strain of F. prausnitzii

A F. prausnitzii strain is isolated from the feces of a healthyvolunteer by microbiological pure culture technique under strictanaerobic condition (5% H₂, 15% CO₂ and 80% N₂) employed in a Coychamber. The routine culture medium for isolation contains following(g/L); yeast extract: 2.5; casitone: 10; glucose: 4.5; sodium chloride:0.9; dipotasium phosphate: 0.45; potassium dihydrogen phosphate: 0.45;ammonium sulfate: 1.32; sodium bicarbonate: 4 g; cysteine: 1; resazurin:0.001; hemin: 0.01. Vitamin mix contains: 10 μg biotin, 10 μg cobalamin,30 μg p-aminobenzoic acid, 50 μg folic acid and 150 μg pyridoxamine.Final concentrations of short-chain fatty acids (SCFA) in the medium are33 mM acetate, 9 mM propionate and 1 mM each of isobutyrate, isovalerateand valerate. All components are added aseptically while the tubes areflushed with CO₂. Heat labile vitamins are filter sterilized with 0.22μm filter and added after the medium is autoclaved to give a finalconcentration of 0.05 μg thiamine ml⁻¹ and 0.05 μg riboflavin ml⁻¹. Thefinal pH of the medium is adjusted with 1N NaOH or 1N HCl to 7.2±0.2.The media is autoclaved at 100 kPa at 121° C. for 15 mins.

Example 3

Alternative Isolation of a Strain of Desulfovibrio piger.

A Desulfovibrio piger strain is isolated from the feces of healthyvolunteer by microbiological pure culture technique under strictanaerobic condition (5% H₂, 15% CO₂ and 80% N₂) employed in a Coychamber. The Postgate medium (PGM) is employed as routine culture mediumfor isolation and cultivation.

The postgate medium contains following (g/L); dipotassium phosphate: 0.5g; ammonium chloride: 1; sodium lactate: 3.5; yeast extract: 1;Ascorbate: 0.1; cysteine: 0.5; sodium chloride: 1; peptone: 10; sodiumsulphate: 1; calcium chloride dehydrate: 1; magnesium sulphate: 2;ferrous sulphate heptahydrate: 0.5. Sodium sulphate, magnesium sulphateheptahydrate, calcium chloride dehydrate are autoclaved separately whileferrous sulfate heptahydrate is filter sterilized 0.22 μm filter andadded after autoclaving and mixing of all components. The final pH ofthe medium is adjusted with 1N NaOH or 1N HCl to 7.2±0.2.

The media is autoclaved at 100 kPa at 121° C. for 15 mins.

Example 4

Genomic Sequencing of Faecalibacterium prausnitzii DSM 32186

The aim of this study was to sequence the genome of the probioticcandidate Faecalibacterium prausnitzii DSM 32186 to identify factorsimportant for metabolic interactions between Faecalibacteriumprausnitzii DSM 32186 and Desulfovibrio piger DSM 32187.

Experimental Procedure and Results

Sequencing and Assembly

A culture of the strain F. prausnitzii DSM 32186 was harvested and DNAwas isolated. The isolated DNA was sequenced on a Pacific Biosciences RSinstrument, at SciLifeLab Uppsala, Sweden. Sequencing generated 73,071reads with an N50 read length of 17,241 base pairs (bp). Sequence readswere assembled using the PacBio SMRTPortal and HGAP version 3 assemblyprotocol (Chin et al., 2013). Default parameters were used except forsetting the estimated genome size to 3 Mbp. Assembly resulted in asingle contig of 2,915,013 bp with a mean coverage of 197. The assemblywas circularized, duplicated trailing ends were trimmed with the AMOSpackage (Treangen et al., 2002) and the start of the chromosome was setto the DnaA gene start codon. To refine the assembly and circularizationmerged ends, the generated circularized chromosome was used as areference in the SMRTPortal Resequence version 1 protocol that mappedthe PacBio reads back to this reference and generates a consensussequence. This consensus sequence was used for further analyses. Initialgene calling and annotation by the NCBI Prokaryotic Genome AnnotationPipeline (Angiuoli et al., 2008) indicated that the assembly containedan unnaturally high number of frameshifted genes (489 out of a total of2,767 genes). Manual inspection of the assembly indicated that theframeshift occurred in homopolymer stretches of Gs or Cs with a lengthof about 6 bp. To mitigate the homopolymer problem, the same DNA samplewas sent for sequencing using the Illumina technology at GATCBiotech.

A total of 7,624,279 paired end reads with a read length of 126 bp wasgenerated with the Illumina Hiseq instrument. Trimmomatic 0.36 (Bolgeret al., 2014) was used for read quality control and filtering of adaptersequences. A total of 6,424,651 high-quality paired-end reads were usedin downstream analysis. The high quality reads were aligned to thePacBio generated consensus sequence with bowtie2 2.2.9 (Langmead andSalzberg, 2012) using default parameters except allowing insert sizelength of 600 bp (−×600). Out of the high quality read pairs 90.2%aligned to the genome concordantly and the overall alignment rate was99.75%. The alignment was supplied to Pilon (Walker et al., 2014) 1.18(https://github.com/broadinstitute/pilon/releases) for correction of theassembly. Pilon made 1074 corrections to the assembly, the vast majoritybeing single insertions of either G or C.

The final assembly of the Faecalibacterium prausnitzii DSM 32186 genomecontained 2,905,188 base pairs and was submitted to NCBI and receivedthe accession number CP015751.

Genome Annotation

The genome sequence was annotated with the NCBI Prokaryotic GenomeAnnotation Pipeline and a total of 2,737 genes were found on thechromosome out of which 2,608 were protein coding genes, 86 RNA genesand 43 pseudogenes. The genome contains 6 complete 5S, 16S and 23Sribosomal genes and 64 tRNA genes.

Unique Genetic Potential Compared to Other Sequenced F. prausnitziiGenomes

The genome of F. prausnitzii DSM 32186 was annotated by the RapidAnnotation using Subsystem Technology (RAST) Server(http://rast.nmpdr.org/) A comparison of the annotation of genes in thesequenced genomes, F. prausnitzii A2-165, SL3/3, KLE1255, L2-6 and M212to F. prausnitzii DSM 32186 identified the following unique functionsare listed in Table 1.

Of special interest is the L-lactate dehydrogenase which is not found inany of the other sequenced F. prausnitzii strains. This protein,A8C61_00370, in DSM 32186 do not have a close homolog in the othersequenced F. prausnitzii genomes and a sequence alignment search withBLAST to the NCBI nr database identifies L-lactate dehydrogenases fromEubacterium, Oribacterium and Roseburia as closely related sequences.The A8C61_00370 L-lactate dehydrogenase has likely been transferred intoF prausnitzii DSM 32186 by horizontal gene transfer since it is indirect proximity to a genomic island identified by IslandViewer 3(Dhillon et al., 2015) online tool(http://www.pathogenomics.sfu.ca/islandviewer/). Experimental data showsthat the DSM 32186 strain produces more lactate during growth which is asubstrate for D piger and therefore supports their interaction.

TABLE 1 Unique gene annotations in F prausnitzii DSM 32186 compared tothe following sequenced F. prausntizii genomes. Bold strain namesspecifies the strain to which DSM 32186 has unique functions. M212:SL3/3Sucrose phosphorylase (EC 2.4.1.7) L-alanine-DL-glutamate epimerase tRNA(5-methylaminomethyl-2-thiouridylate)-methyltransferase (EC 2.1.1.61)Beta-lactamase (EC 3.5.2.6) A2-165:L2-6 Serine hydroxymethyltransferase(EC 2.1.2.1) CRISPR-associated helicase Cas3 CRISPR-associated protein,Cas5e family CRISPR-associated protein, Cse1 family CRISPR-associatedprotein, Cse2 family CRISPR-associated protein, Cse3 familyCRISPR-associated protein, Cse4 family Ribonucleotide reductase of classIa (aerobic), alpha subunit (EC 1.17.4.1) Ribonucleotide reductase ofclass Ia (aerobic), beta subunit (EC 1.17.4.1) DNA primase/helicase,phage-associated Autolysis response regulater LytR Choline bindingprotein A A2-165:M212 Putative predicted metal-dependent hydrolaseTranscriptional regulator, MerR family A2-165:SL3/3UDP-N-acetylglucosamine 2-epimerase (EC 5.1.3.14) KLE1255:L2-6L-asparaginase I, cytoplasmic (EC 3.5.1.1) Alpha-galactosidase (EC3.2.1.22) PTS system, maltose and glucose-specific IIB component (EC2.7.1.69) PTS system, maltose and glucose-specific IIC component (EC2.7.1.69) Acetaldehyde dehydrogenase (EC 1.2.1.10) tRNA S(4)U4-thiouridine synthase (former ThiI) KLE1255:M212 capsularpolysaccharide biosynthesis protein Multidrug and toxin extrusion (MATE)family efflux pump YdhE/NorM, homolog KLE1255:SL3/3 Adenine-specificmethyltransferase (EC 2.1.1.72) Type III restriction-modification systemmethylation subunit (EC 2.1.1.72) RNA polymerase sigma-54 factor RpoNA2-165:KLE1255 putative esterase Manganese-dependent protein-tyrosinephosphatase (EC 3.1.3.48) Hydrolase (HAD superfamily) in cluster withDUF1447 Putative DNA-binding protein in cluster with Type Irestriction-modification system RelE/StbE replicon stabilization toxinL2-6:M212:SL3/3 Phosphoglycerate mutase family A2-165:M212:SL3/3Galactose permease Anion permease ArsB/NhaD-like KLE1255:L2-6:M212Potassium efflux system KefA protein KLE1255:L2-6:SL3/3 GalactosideO-acetyltransferase (EC 2.3.1.18) Anti-sigma B factor antagonist RsbVKLE1255:M212:SL3/3 Chorismate mutase I (EC 5.4.99.5)N-Acetyl-D-glucosamine ABC transport system, permease protein 1 Pectindegradation protein KdgF Glycerol-3-phosphate ABC transporter,periplasmic glycerol-3-phosphate-binding protein (TC 3.A.1.1.3) Innermembrane protein translocase component YidC, long form Thermostablecarboxypeptidase 1 (EC 3.4.17.19) VapB protein (antitoxin to VapC)A2-165:KLE1255:L2-6 Bipolar DNA helicase HerA Ribosome-associated heatshock protein implicated in the recycling of the 50S subunit (S4paralog) Ubiquinone/menaquinone biosynthesis methyltransferase UbiE (EC2.1.1.—) tRNA-specific 2-thiouridylase MnmA A2-165:KLE1255:M212 Phagetail fiber protein A2-165:L2-6:M212:SL3/3 Phage major tail protein NADHdehydrogenase (EC 1.6.99.3) KLE1255:L2-6:M212:SL3/3 Cellobiosephosphotransferase system YdjC-like protein Lactose and galactosepermease, GPH translocator family Pseudouridine-5&#39; phosphatase (EC3.1.3.—) Duplicated ATPase component MtsB of energizing module ofmethionine-regulated ECF transporter Substrate-specific component MtsAof methionine-regulated ECF transporter Transmembrane component MtsC ofenergizing module of methionine-regulated ECF transporter Phage majorcapsid protein Aminopeptidase C (EC 3.4.22.40) Rrf2 familytranscriptional regulator, group III YoeB toxin proteinA2-165:KLE1255:M212:SL3/3 Oligo-1,6-glucosidase (EC 3.2.1.10)Co-activator of prophage gene expression IbrB TTE0858 repliconstabilization protein (antitoxin to TTE0859)A2-165:KLE1255:L2-6:M212:SL3/3 Maltodextrin glucosidase (EC 3.2.1.20)Maltose O-acetyltransferase (EC 2.3.1.79) L-lactate dehydrogenase (EC1.1.1.27) Maltose/maltodextrin ABC transporter, substrate bindingperiplasmic protein MalE Choline kinase (EC 2.7.1.32) Choline permeaseLicB Cholinephosphate cytidylyltransferase (EC 2.7.7.15)Lipopolysaccharide cholinephosphotransferase LicD1 (EC 2.7.8.—)Alpha-L-Rha alpha-1,3-L-rhamnosyltransferase (EC 2.4.1.—)Single-stranded exonuclease associated with Rad50/Mre11 complex DNAmismatch repair endonuclease MutH Very-short-patch mismatch repairendonuclease (G-T specific) Mg(2+) transport ATPase protein CCo-activator of prophage gene expression IbrA Phage terminase smallsubunit Phage replication initiation protein tRNA-Ala tRNA-Arg tRNA-AsntRNA-Asp tRNA-Cys tRNA-Gln tRNA-Glu tRNA-Gly tRNA-His tRNA-Ile tRNA-LeutRNA-Lys tRNA-Met tRNA-Phe tRNA-Pro tRNA-Ser tRNA-Thr tRNA-Trp tRNA-TyrtRNA-Val Cytochrome c-type biogenesis protein DsbD, protein-disulfidereductase (EC 1.8.1.8) Glycine betaine transporter OpuD HtrAprotease/chaperone protein Beta-lactamase class C and other penicillinbinding proteins L2-6 Phosphoribosylformimino-5-aminoimidazolecarboxamide ribotide isomerase (EC 5.3.1.16) ATPphosphoribosyltransferase (EC 2.4.2.17) ATP phosphoribosyltransferaseregulatory subunit (EC 2.4.2.17) Histidinol dehydrogenase (EC 1.1.1.23)Histidinol-phosphate aminotransferase (EC 2.6.1.9) Imidazole glycerolphosphate synthase amidotransferase subunit (EC 2.4.2.—) Imidazoleglycerol phosphate synthase cyclase subunit (EC 4.1.3.—)Imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19) Cellobiosephosphorylase (EC 2.4.1.—) Citrate synthase (si) (EC 2.3.3.1)N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) NAD(P)HX epimeraseAlcohol dehydrogenase (EC 1.1.1.1) Isocitrate dehydrogenase [NADP] (EC1.1.1.42) Recombination inhibitory protein MutS2 Phosphate:acyl-ACPacyltransferase PlsX Repressor CsoR of the copZA operon TRAP-typeC4-dicarboxylate transport system, small permease component M212Putative tRNA-m1A22 methylase Phage portal protein SL3/3 DinG familyATP-dependent helicase CPE1197 Alpha-aspartyl dipeptidase Peptidase E(EC 3.4.13.21) A2-165 Beta-glucosidase (EC 3.2.1.21) Beta-glucoside bgloperon antiterminator, BglG family Maltose/maltodextrin ABC transporter,permease protein MalF Maltose/maltodextrin ABC transporter, permeaseprotein MalG Glycerol kinase (EC 2.7.1.30) Tyrosine-protein kinase EpsD(EC 2.7.10.2) Tyrosine-protein kinase transmembrane modulator EpsCAlpha-D-GlcNAc alpha-1,2-L-rhamnosyltransferase (EC 2.4.1.—) TRAP-typetransport system, small permease component, predicted N-acetylneuraminate transporter Rod shape-determining protein RodARNA-binding protein Jag 3,4-dihydroxy-2-butanone 4-phosphate synthase(EC 4.1.99.12) 5-amino-6-(5-phosphoribosylamino)uracil reductase (EC1.1.1.193) 6,7-dimethyl-8-ribityllumazine synthase (EC 2.5.1.78)Diaminohydroxyphosphoribosylaminopyrimidine deaminase (EC 3.5.4.26) GTPcyclohydrolase II (EC 3.5.4.25) Riboflavin synthaseeubacterial/eukaryotic (EC 2.5.1.9) Chromosome partition protein smc(2E,6E)-farnesyl diphosphate synthase (EC 2.5.1.10)Dimethylallyltransferase (EC 2.5.1.1) Octaprenyl diphosphate synthase(EC 2.5.1.90) Lipid carrier: UDP-N-acetylgalactosaminyltransferase (EC2.4.1.—) Ferroxidase (EC 1.16.3.1) Iron-binding ferritin-likeantioxidant protein Non-specific DNA-binding protein Dps Superoxidereductase (EC 1.15.1.2) transcriptional regulator, Crp/Fnr familyKLE1255 Undecaprenyl-phosphate galactosephosphotransferase (EC 2.7.8.6)YafQ toxin protein Formate dehydrogenase chain D (EC 1.2.1.2)

Example 5

Evaluation of the Synergistic Effects

To evaluate the synergistic effects a co-culture from Example 1 wasused. As an alternative the F. prausnitzii strain FBT-22 (DSM 32186)from Example 2 was co-cultured with the D. piger strain FBT-23 (DSM32187) from Example 3 in postgate medium under strict anaerobicconditions.

The postgate medium contains following (g/L); dipotassium phosphate: 0.5g; ammonium chloride: 1; sodium lactate: 3.5; yeast extract: 1;Ascorbate: 0.1; cysteine: 0.5; sodium chloride: 1; peptone: 10; sodiumsulphate: 1; calcium chloride dehydrate: 1; magnesium sulphate: 2;ferrous sulphate heptahydrate: 0.5. Sodium sulphate, magnesium sulphateheptahydrate, calcium chloride dehydrate were autoclaved separatelywhile ferrous sulfate heptahydrate was filter sterilized 0.22 μm filterand added after autoclaving and mixing of all components. The final pHof the medium was adjusted with 1N NaOH or 1N HCl to 7.2±0.2.

The media was autoclaved at 100 kPa at 121° C. for 15 mins.

This resulted in the following data (Table 2), illustrating thesynergistic effects of the strains on butyrate production.

TABLE 2 Fold change in fatty acid profile Formate Acetate ButyrateLactate Blank PGM 1 1 1 1 D. piger (D.p) 0.8 118.5 1.1 0 FBT-23 F.prausnitzii (F.p) 0.9 1.4 7.1 1.1 FBT-22 F.p FBT-22 + D.p 0.8 122.7 25.20 FBT-23

Example 6

Manufacture of Product Containing Both Strains

In the present study we separately grew the F. prausnitzii strain FBT-22(DSM 32186) and the D. piger strain FBT-23 (DSM 32187) in PGM understrict anaerobic conditions.

The PGM contains (g/L); dipotassium phosphate: 0.5 g; ammonium chloride:1; sodium lactate: 3.5; yeast extract: 1; Ascorbate: 0.1; cysteine: 0.5;sodium chloride: 1; peptone: 10; sodium sulphate: 1; calcium chloridedehydrate: 1; magnesium sulphate: 2; ferrous sulphate heptahydrate: 0.5.Sodium sulphate, magnesium sulphate heptahydrate, calcium chloridedehydrate were autoclaved separately while ferrous sulfate heptahydratewas filter sterilized 0.22 μm filter and added after autoclaving andmixing of all components. The final pH of the medium was adjusted with1N NaOH or 1N HCl to 7.2±0.2.

The media was autoclaved at 100 kPa at 121° C. for 15 mins.

After growing the bacteria into stationary phase, the cells where washedin distilled water and then concentrated using a centrifuge forsensitive material. The resulting slurry of each strain was measured inaliquots containing 1 E+9 CFU and then mixed with each other in ratio1:1. The product was then preserved in 20% glycerol and kept at −80° C.

Example 7

Metabolic Profiles of Type Strain of Faecalibacterium prausnitzii A2-165(DSM 17677) and Faecalibacterium prausnitzii FBT-22 (DSM 32186) inPhysiologically Relevant Medium LYBHI

The detailed metabolic profiles of Faecalibacterium prausnitzii strainsare depicted in FIG. 1 and FIG. 2. In aforementioned figures it isobvious that on glucose fermentation F. prausnitzii can produce butyrateas the major SCFA and consumes acetate. Additionally these bacteriaproduce lactate, formate and acetate.

The comparative metabolic profiles of type strain of Faecalibacteriumprausnitzii A2-165 (DSM 17677) and Faecalibacterium prausnitzii FBT-22(DSM 32186) in physiologically relevant medium LYBHI reveals that thesetwo bacteria are metabolically different (Fold change of SCFA is shownin Table 3). The comparative glucose consumption and butyrate productionwas almost similar, however, the two strains differs in lactateproduction (FIG. 3)

Composition of physiologically relevant growth medium LYBHI: LYBHImedium (brain-heart infusion medium supplemented with 0.5% yeastextract) (Oxoid, UK) supplemented with 1 mg/ml cellobiose (Sigma-AldrichChemie GmbH, Buchs, Switzerland), 1 mg/ml maltose (Sigma-Aldrich), and0.5 mg/ml cysteine (Sigma-Aldrich).

The complete carbon and electron balances are presented in Table 4.

Example 8

Metabolic Profiles of Mono-Culture and Co-Culture of Type StrainFaecalibacterium prausnitzii A2-165 (DSM 17677) and Desulfovibrio pigerStrain FBT-23 (DSM 32187) in LYBHI Medium

The synergistic effect of type strain Faecalibacterium prausnitziiA2-165 and Desulfovibrio piger strain FBT-23 was evaluated in LYBHImedium (composition described in Example 7).

As shown in FIG. 2, lactate produced by F. prausnitzii used as electrondonor by D. piger and vice versa acetate generated by D. piger can beutilized by F. prausnitzii for butyrate production.

In LYBHI medium under the co-culture conditions butyrate production was1.5 fold increased and lactate production was 2.3 fold decreased (FIG.4, table 3). This indicates beneficial effect of co-culture/crossfeeding i.e. increased butyrate production.

The complete carbon and electron balances are presented in Table 4.

Example 9

Metabolic Profiles of Mono-Culture and Co-Culture of Faecalibacteriumprausnitzii FBT-22 (DSM 32186) and Desulfovibrio piger Strain FBT-23(DSM 32187) in LYBHI Medium

The synergistic effect of Faecalibacterium prausnitzii FBT-22 andDesulfovibrio piger strain FBT-23 was evaluated in LYBHI medium(composition described in Example 7).

In LYBHI medium, under co-culture conditions the butyrate production was2.1 fold increased and lactate accumulation was 2 fold decreased (FIG.5, Table 3). This shows that there is a cross feeding and synergisticeffect.

The complete carbon and electron balances are presented in Table 4.

TABLE 3 Fold change SCFA of Faecalibacterium prausnitzii isolate andtype strain A2-165 in physiologically relevant growth medium Brain HeartInfusion (LYBHI). The data illustrates the synergistic effects of thedifferent strains on butyrate production. Butyrate Acetate Lactate BlankLYBHI medium 1.0 1.0 1.0 D. piger 1.0 3.5 0.4 FBT-23 (D.p) F.prausnitzii 4.2 0.0 14.1 FBT-22 (F.p) F.p + D.p 7.5 1.8 7.1 F.prausnitzii type strain 5.0 0.0 8.8 (F.p A2165) F.p A2-165 + D.p 6.8 0.03.8

TABLE 4 carbon and electron balance of mono-culture, co-cultures ofFaecalibacterium prausnitzii FBT-22 (DSM 32186), Faecalibacteriumprausnitzii A2-165 (DSM 17677) and Desulfovibrio piger strain FBT-23(DSM 32187) Recoveries of Carbon and Electrons Growth medium, bacterialstrains % and their respective combinations Butyrate Lactate FormateAcetate Glucose Recovery Number of Carbon (C)/mol 4 3 1 2 6 Number ofElectrons (e⁻)/mol 20 12 2 8 24 BHI medium F. prausnitzii A2-165 (DSM17677) mM 9.9 29.8 0.0 0.0 −14.6 mM C 40 89 0 0 −88 141 mM e⁻ 198 357 00 −351 158 F. prausnitzii A2-165 (DSM 17677) + mM 14.6 12.6 3.2 0.0−15.0 D. piger FBT-23 (DSM 32187) mM C 58 38 3 0 −90 110 mM e⁻ 292 151 60 −360 125 F. prausnitzii FBT-22 (DSM 32186) mM 7.8 48.3 0.0 0.0 −15.0mM C 31 145 0 0 −90 196 mM e⁻ 156 579 0 0 −360 204 F. prausnitzii FBT-22(DSM 32186) + mM 16.5 24.1 0.0 0.0 −15.0 D. piger FBT-23 (DSM 32187) mMC 66 72 0 2 −90 151 mM e⁻ 329 289 0 10 −360 169

The invention claimed is:
 1. A method of increasing butyrate productionby F. prausnitzii and/or increasing the levels of F. prausnitzii in thegut of a subject, the method comprising administering to a subjecthaving reduced butyrate production and/or reduced or low levels ofFaecalibacterium prausnitzii in the gut a strain of F. prausnitzii and astrain of Desulfovibrio piger, wherein the D. piger strain has one ormore of the characteristics of: (i) producing acetate, (ii) consuminglactate and (iii) being an electron acceptor, thereby increasingbutyrate production by F. prausnitzii and/or increasing the levels of F.prausnitzii in the gut of the subject, wherein the increase in butyrateproduction and/or the increase in levels of F. prausnitzii is asynergistic effect of administering the strain of F. prausnitzii and thestrain of D. piger.
 2. The method of claim 1, wherein said strains areadministered together in combination, or sequentially, or separately. 3.The method of claim 1, wherein said D. piger strain has all of thecharacteristics (i) to (iii).
 4. The method of claim 1, wherein saidstrain of F. prausnitzii (i) produces butyrate, and optionally: (ii)consumes acetate, (iii) produces extracellular electrons and/or (iv)produces lactate, or any combination thereof.
 5. The method of claim 4,wherein said strain of F. prausnitzii has all of the characteristics (i)to (iv) and/or said strain of D. piger has all of the characteristics(i) to (iii).
 6. The method of claim 1, wherein said strain of F.prausnitzii comprises a L-lactate dehydrogenase gene.
 7. The method ofclaim 1, wherein the strain of F. prausnitzii is DSM 32186 and/or thestrain of D. piger is DSM
 32187. 8. The method of claim 1, whereinadministering to the subject produces an increased growth andcolonization of F. prausnitzii bacteria in the subject'sgastrointestinal tract.
 9. The method of claim 1, wherein the reducedbutyrate production or reduced or low levels of F. prausnitzii in thegut of the subject results in a disease selected from the groupconsisting of type 2 diabetes, gestational diabetes, obesity, gout,pouchitis, chronic kidney disease, psoriasis, inflammatory boweldisease, irritable bowel syndrome, and constipation.
 10. The method ofclaim 1, wherein reduced butyrate production or reduced or low levels ofF. prausnitzii in the gut of the subject results in type 2 diabetes orgestational diabetes.
 11. The method of claim 1, wherein the increase inbutyrate production is an increase of at least 1.36 fold.